Gene Expression Analysis Identifies Potential Biomarkers of Neurofibromatosis Type 1 Including Adrenomedullin

Division of Experimental Hematology and Cancer Biology, Cincinnati Children's Hospital Medical Center, Cincinnati, Ohio 45229, USA.
Clinical Cancer Research (Impact Factor: 8.72). 10/2010; 16(20):5048-57. DOI: 10.1158/1078-0432.CCR-10-0613
Source: PubMed


Plexiform neurofibromas (pNF) are Schwann cell tumors found in a third of individuals with neurofibromatosis type 1 (NF1). pNF can undergo transformation to malignant peripheral nerve sheath tumors (MPNST). There are no identified serum biomarkers of pNF tumor burden or transformation to MPNST. Serum biomarkers would be useful to verify NF1 diagnosis, monitor tumor burden, and/or detect transformation.
We used microarray gene expression analysis to define 92 genes that encode putative secreted proteins in neurofibroma Schwann cells, neurofibromas, and MPNST. We validated differential expression by quantitative reverse transcription-PCR, Western blotting, and ELISA assays in cell conditioned medium and control and NF1 patient sera.
Of 13 candidate genes evaluated, only adrenomedullin (ADM) was confirmed as differentially expressed and elevated in serum of NF1 patients. ADM protein concentrati on was further elevated in serum of a small sampling of NF1 patients with MPNST. MPNST cell conditioned medium, containing ADM and hepatocyte growth factor, stimulated MPNST migration and endothelial cell proliferation.
Thus, microarray analysis identifies potential serum biomarkers for disease, and ADM is a serum biomarker of NF1. ADM serum levels do not seem to correlate with the presence of pNFs but may be a biomarker of transformation to MPNST.

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    • "Recently, two studies identified MIA and ADM as potential NF1 tumor markers in cohorts of 42 and 32 patients, respectively [21,23]. There was also a trend towards correlation between ADM and MPNST, although the MPNST group was too small to show significance (n = 5). "
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    ABSTRACT: Background Neurofibromatosis type 1 (NF1) is a hereditary tumor syndrome characterized by the development of benign nerve-sheath tumors, which transform to malignant peripheral nerve-sheath tumors (MPNST) in about 8 to 13% of patients with NF1. MPNST are invasive sarcomas with extremely poor prognosis, and their development may correlate with internal tumor load of patients with NF1. Because early identification of patients with NF1 at risk for developing MPNST should improve their clinical outcome, the aim of this study was to identify serum biomarkers for tumor progression in NF1, and to analyze their correlation with tumor type and internal tumor load. Methods We selected candidate biomarkers for NF1 by manually mining published data sources, and conducted a systematic screen of 56 candidate serum biomarkers using customized antibody arrays. Serum from 104 patients with NF1 with and without MPNST, and from 41 healthy control subjects, was analyzed. Statistical analysis was performed using the non-parametric Mann–Whitney U-test, followed by Bonferroni correction. Results Our analysis identified four markers (epidermal growth factor receptor, interferon-γ, interleukin-6, and tumor necrosis factor-α) for which significantly different serum concentrations were seen in patients with NF1 compared with healthy controls. Two markers (insulin-like growth factor binding protein 1 (IGFBP1) and regulated upon activation, normal T-cell expressed and secreted (RANTES)) showed significantly higher concentrations in patients with NF1 and MPNST compared with patients with NF1 without MPNST. A correlation with internal tumor load was found for IGFBP1. Conclusion Our study identified two serum markers with potential for early detection of patients with NF1 at risk for developing MPNST, and four markers that could distinguish between patients with NF1 and healthy subjects. Such markers may be useful as diagnostic tools to support the diagnosis of NF1 and for timely identification of MPNST. Moreover, the data suggest that there is a systemic increase in inflammatory cytokines independently of tumor load in patients with NF1.
    Full-text · Article · Apr 2013 · BMC Medicine
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    ABSTRACT: p14(ARF), p15(INK4b), and p16(INK4a) are tumor suppressor genes that are located closely at 9p21 and are often coinactivated by genetic or epigenetic alterations. Malignant peripheral nerve sheath tumor (MPNST) is a rare sarcoma with poor prognosis. However, the prognostic implications of inactivation of p14(ARF), p15(INK4b), and p16(INK4a) in MPNSTs have not been adequately investigated. Here we carried out a genetic, epigenetic, and expression analysis of p14(ARF), p15(INK4b), and p16(INK4a), and clarified the prognostic significance of their inactivation in MPNSTs. p14(ARF), p15(INK4b), and p16(INK4a) protein expressions were assessed by immunohistochemistry in 129 formalin-fixed samples of MPNST including 85 primary tumors. Thirty-nine samples, for which frozen material was available, were also investigated by Western blotting and quantitative reverse transcription PCR (RT-PCR) to detect p14(ARF), p15(INK4b), and p16(INK4a) protein and mRNA expression, and by multiplex real-time PCR, PCR single strand conformation polymorphism and methylation-specific PCR to detect p14(ARF), p15(INK4b), and p16(INK4a) gene alterations. Immunohistochemically decreased expressions of p14(ARF), p15(INK4b), and p16(INK4a) were observed in 48%, 54%, and 49% of primary MPNSTs, respectively, and were significantly correlated with their concordant mRNA levels. As for gene alterations, homozygous deletion of CDKN2A was detected in one third of the cases. Inactivation of p14(ARF) and p16(INK4a) was associated with poor prognosis by both univariate and multivariate analyses. Furthermore, cases with inactivation of all p14(ARF), p15(INK4b), and p16(INK4a) genes showed the worst prognosis in a combined prognostic assessment. A comprehensive analysis of p14(ARF), p15(INK4b), and p16(INK4a) inactivation status provides useful prognostic information in MPNSTs.
    No preview · Article · Jun 2011 · Clinical Cancer Research
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